The present invention relates to a compound capable of alkylating a gene, an alkylating agent using the compound, a method for controlling the expression of the gene by using the alkylating agent, and a pharmaceutical composition containing the compound.
The rapid progress in molecular biology sequentially elucidates that the etiology of various diseases including cancer lies in DNA mutation. Furthermore, it is believed that the human genome project will complete the determination of the whole nucleotide sequence of human DNA within two or three years. Accordingly, it is increasingly expected the development of therapeutic methods of these diseases based on molecular-level findings. However, the practical application of these approaches is barred by such a serious barrier that not any general technology has been established for regulating gene expression extracellularly. In recent years, molecules binding to specific nucleotide sequences have been designed on the basis Of the recognition of DNA molecules by antibiotics such as distamycin. Hence, it can be said that gene expression is now possibly under regulation.
X-ray crystallography and NMR have elucidated that crescent-shape antibiotics such as distamycin and netropsin bind to the minor groove of DNA via hydrogen bonds between adenine (sometimes abbreviated as A hereinbelow) and thymine (sometimes abbreviated as T hereinbelow) in a DNA sequence with abundant A-T base pairs. Various molecules have been synthetically produced by utilizing the recognition. It has been speculated so far that the antibiotics will acquire an ability to recognize guanine-cytosine base pairs (G-C; guanine is sometimes abbreviated as G hereinbelow; cytosine is sometimes abbreviated as C hereinbelow), when the pyrrole ring is modified into imidazole [M. L. Kopla, C. Yoon, D. Goodsell, P. Pjura, R. E. Dickerson, Proc. Natl. Acad. Sci. USA, 82, 1376 (1985); J. W. Lown, K. Krowickl, U. G. Bhat, A. Skorobogaty, B. Ward, J. C. Dabrowiak, Biochemistry, 25, 7408 (1986)]. Practically, it never has been so simple. Hence, numerous intriguing experimental results have been reported.
Wemmer et al. examined distamycin bound DNA in detail by NMR. Consequently, they verified that two disks of distamycin can be stacked in the DNA miner group, where it has been believed that only one disk thereof can be placed [J. G. Pelton, D. E. Wemmer, Proc. Natl. Acad. Sci. USA, 86, 5723 (1989)]. Focusing attention to the binding mode by which the intriguing results hitherto can be explained, Dervan et al. demonstrated that the nucleotide sequence of a double-stranded DNA can be recognized by a polyamide comprising a pair of methylpyrrole (sometimes abbreviated as Py hereinbelow) and methylimidazole (sometimes abbreviated as Im hereinbelow) in an anti-parallel orientation.
More specifically, they introduced a general rule that Py-Im recognizes C-G base pair; Im-Py recognizes G-C base pair; and Py-Py recognizes A-T or T-A base pair [S. White, E. E. Baird, P. B. Dervan, Chemistry and Biology, 4, 569 (1997)]. Various polyamides in hair-pin structures or cyclic polyamides have been synthetically produced, so as to introduce a covalent bond in such pairs to prevent the entropy loss during binding, thereby generating stronger binding and higher recognition potency. It is also elucidated that a hair-pin structure with a xcex3-linker represented by the formula xe2x80x94NHCH2CH2CH2COxe2x80x94 particularly exerts excellent binding and recognition potencies. The structure of a complex thereof with DNA is also determined [R. P. L. de Clairac, B. H. Geierstanger, M. Marksich, P. B. Dervan, D. E. Wemmer, J. Am. Chem. Soc., 119, 7909 (1997)].
Hair-pin polyamides just composed of pyrrole and imidazole can recognize base pairs up to 7 [J. M. Turner, E. F. Baird, P. B. Dervan, ibid., 119, 7636 (1997)]. When a xcex2-alanine pair recognizing A-T sequence is introduced into a homo-dimer system, 11 base pairs can be recognized [S. E. Swalley, E. E. Baird, D. B. Dervan, Chem. Eur. J., 3, 1600 (1997)].
Furthermore, Dervan, Gottesfeld et al. designed a polyamide capable of binding to the fourth finger in the recognition sequence of one of Zn finger proteins, namely TFIIIA, in a manner antagonistic against a minor group of sequences to potentially bind to the fourth finger and then revealed that the expression of 5S RNA could be regulated selectively at in vitro experiments [J. M. Gottesfeld, L. Neely, J. W. Trauger, E. E. Baird, P. B. Dervan, Nature, 387, 202 (1997)]. Additionally, they simultaneously demonstrated that the polyamide permeated into nuclei at in vitro experiments.
Recent attention has been drawn toward oligonucleotides and peptide nucleic acids (PNA), as tools for gene regulation. However, these molecules suffered lower cell permeability. Taking account of significant cell permeability of methylpyrrole-methylimidazole polyamides, these molecules are promising compounds as a molecule regulating gene expression, a powerful tool in molecular biology, and a human medicine.
Conventional polyamides of Py-Im series never bind to gene via a covalent bond but only recognize in the minor groove hydrogen bonds between base pairs.
The present invention provides a compound recognizing the minor group of hydrogen bonds between base pairs and being capable of forming a covalent bond with bases. The inventive compound can recognize a specific nucleotide sequence and can strongly bind to adjacent bases via covalent bonding, to regulate the expression of a DNA with the nucleotide sequence.
The invention relates to a compound represented by the general formula I: 
wherein
R represents a lower amyl group or a polyamide group;
and X represents nitrogen or CH;
wherein R represents a lower alkyl group or a polyamide group;
and X represents nitrogen or CH.
The invention relates to an alkylating agent of gene, comprising a compound represented by the general formula I. More specifically, the inventive alkylating agent recognizes a specific nucleotide sequence in a gene, such as W-W-V wherein V represents A or G; and W represents A or T or U (abbreviation of uracil), or G-W-V wherein V represents A or G; and W represents A or T or U, thereby selectively alkylating the sequence.
Furthermore, the invention relates to a method for regulating the expression of a gene region including a specific nucleotide sequence, by using the alkylating agent.
Still furthermore, the invention relates to a pharmaceutical composition for the treatment and prophylaxis of various gene-inducible diseases, the composition containing a compound represented by the general formula I.